LOCUS       sequence-486897-        6406 bp    DNA     circular SYN 23-SEP-2025
DEFINITION  Mammalian Expression of 3BoxB.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6406)
  TITLE     Izaurralde lab plasmids
REFERENCE   2  (bases 1 to 6406)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Sep 23, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"
COMMENT     Sequence Label: pEGFP-N3-FLuc-3BoxB_O
FEATURES             Location/Qualifiers
     source          1..6406
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        61..364
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        365..568
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     519..539
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     regulatory      600..609
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             606..2255
                     /codon_start=1
                     /gene="luc+"
                     /product="firefly luciferase"
                     /label=luciferase
                     /note="enhanced luc+ version of the luciferase gene"
                     /translation="MEYAKNIKKGPAPFYPLEDGTAGEQLHKAMKRYALVPGTIAFTDA
                     HIEVDITYAEYFEMSVRLAEAMKRYGLNTNHRIVVCSENSLQFFMPVLGALFIGVAVAP
                     ANDIYNERELLNSMGISQPTVVFVSKKGLQKILNVQKKLPIIQKIIIMDSKTDYQGFQS
                     MYTFVTSHLPPGFNEYDFVPESFDRDKTIALIMNSSGSTGLPKGVALPHRTACVRFSHA
                     RDPIFGNQIIPDTAILSVVPFHHGFGMFTTLGYLICGFRVVLMYRFEEELFLRSLQDYK
                     IQSALLVPTLFSFFAKSTLIDKYDLSNLHEIASGGAPLSKEVGEAVAKRFHLPGIRQGY
                     GLTETTSAILITPEGDDKPGAVGKVVPFFEAKVVDLDTGKTLGVNQRGELCVRGPMIMS
                     GYVNNPEATNALIDKDGWLHSGDIAYWDEDEHFFIVDRLKSLIKYKGYQVAPAELESIL
                     LQHPNIFDAGVAGLPDDDAGELPAAVVVLEHGKTMTEKEIVDYVASQVTTAKKLRGGVV
                     FVDEVPKGLTGKLDARKIREILIKAKKGGKIAV"
     primer_bind     complement(672..690)
                     /label=LucNrev
                     /note="Firefly luciferase, reverse primer"
     regulatory      2265..2274
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(2271..2273,2274..2276,2277..2990)
                     /codon_start=1
                     /product="the original enhanced GFP (Yang et al., 1996)"
                     /label=EGFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     CDS             join(2271..2273,2274..2276,2277..2987)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(2316..2337)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(2577..2596)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     2924..2945
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     protein_bind    3045..3063
                     /label=boxB
                     /bound_moiety="lambda-N peptide"
                     /note="lambda N peptide binding site consisting of an RNA 
                     hairpin within the N-utliization (nut) site of the lambda 
                     bacteriophage transcript (Tan and Frankel, 1995)"
     protein_bind    3089..3107
                     /label=boxB
                     /bound_moiety="lambda-N peptide"
                     /note="lambda N peptide binding site consisting of an RNA 
                     hairpin within the N-utliization (nut) site of the lambda 
                     bacteriophage transcript (Tan and Frankel, 1995)"
     protein_bind    3113..3131
                     /label=boxB
                     /bound_moiety="lambda-N peptide"
                     /note="lambda N peptide binding site consisting of an RNA 
                     hairpin within the N-utliization (nut) site of the lambda 
                     bacteriophage transcript (Tan and Frankel, 1995)"
     polyA_signal    3266..3387
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3303..3322)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     3357..3376
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      complement(3394..3849)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3531..3552)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     3743..3762
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     promoter        3876..3980
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(4005..4025)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        4049..4259
                     /label=SV40 promoter
                     /note="SV40 early promoter"
     promoter        4071..4267
                     /label=SV40 promoter
                     /note="SV40 early promoter"
     rep_origin      4118..4253
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     CDS             4302..5096
                     /codon_start=1
                     /product="aminoglycoside phosphotransferase"
                     /label=NeoR/KanR
                     /note="confers resistance to neomycin, kanamycin, and G418 
                     (Geneticin(R))"
                     /translation="MIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGRP
                     VLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDLLS
                     SHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDEEHQ
                     GLAPAELFARLKASMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
                     LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     CDS             4302..5096
                     /codon_start=1
                     /gene="aph(3')-II (or nptII)"
                     /product="aminoglycoside phosphotransferase from Tn5"
                     /label=NeoR/KanR
                     /note="confers resistance to neomycin, kanamycin, and G418 
                     (Geneticin(R))"
                     /translation="MIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGRP
                     VLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDLLS
                     SHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDEEHQ
                     GLAPAELFARLKASMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
                     LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     primer_bind     complement(4356..4375)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     4966..4985
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     complement(5284..5303)
                     /label=TK-pA-R
                     /note="Thymidine kinase polyA, reverse primer"
     polyA_signal    5328..5375
                     /label=HSV TK poly(A) signal
                     /note="herpes simplex virus thymidine kinase 
                     polyadenylation signal (Cole and Stacy, 1985)"
     rep_origin      5704..6292
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6193..6212
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 tagttattaa tagtaatcaa ttacggggtc attagttcat agcccatata tggagttccg
       61 cgttacataa cttacggtaa atggcccgcc tggctgaccg cccaacgacc cccgcccatt
      121 gacgtcaata atgacgtatg ttcccatagt aacgccaata gggactttcc attgacgtca
      181 atgggtggag tatttacggt aaactgccca cttggcagta catcaagtgt atcatatgcc
      241 aagtacgccc cctattgacg tcaatgacgg taaatggccc gcctggcatt atgcccagta
      301 catgacctta tgggactttc ctacttggca gtacatctac gtattagtca tcgctattac
      361 catggtgatg cggttttggc agtacatcaa tgggcgtgga tagcggtttg actcacgggg
      421 atttccaagt ctccacccca ttgacgtcaa tgggagtttg ttttggcacc aaaatcaacg
      481 ggactttcca aaatgtcgta acaactccgc cccattgacg caaatgggcg gtaggcgtgt
      541 acggtgggag gtctatataa gcagagctgg tttagtgaac cgtcagatcc gctagcacag
      601 ccaccatgga atacgccaaa aacataaaga aaggcccggc gccattctat ccgctggaag
      661 atggaaccgc tggagagcaa ctgcataagg ctatgaagag atacgccctg gttcctggaa
      721 caattgcttt tacagatgca catatcgagg tggacatcac ttacgctgag tacttcgaaa
      781 tgtccgttcg gttggcagaa gctatgaaac gatatgggct gaatacaaat cacagaatcg
      841 tcgtatgcag tgaaaactct cttcaattct ttatgccggt gttgggcgcg ttatttatcg
      901 gagttgcagt tgcgcccgcg aacgacattt ataatgaacg tgaattgctc aacagtatgg
      961 gcatttcgca gcctaccgtg gtgttcgttt ccaaaaaggg gttgcaaaaa attttgaacg
     1021 tgcaaaaaaa gctcccaatc atccaaaaaa ttattatcat ggattctaaa acggattacc
     1081 agggatttca gtcgatgtac acgttcgtca catctcatct acctcccggt tttaatgaat
     1141 acgattttgt gccagagtcc ttcgataggg acaagacaat tgcactgatc atgaactcct
     1201 ctggatctac tggtctgcct aaaggtgtcg ctctgcctca tagaactgcc tgcgtgagat
     1261 tctcgcatgc cagagatcct atttttggca atcaaatcat tccggatact gcgattttaa
     1321 gtgttgttcc attccatcac ggttttggaa tgtttactac actcggatat ttgatatgtg
     1381 gatttcgagt cgtcttaatg tatagatttg aagaagagct gtttctgagg agccttcagg
     1441 attacaagat tcaaagtgcg ctgctggtgc caaccctatt ctccttcttc gccaaaagca
     1501 ctctgattga caaatacgat ttatctaatt tacacgaaat tgcttctggt ggcgctcccc
     1561 tctctaagga agtcggggaa gcggttgcca agaggttcca tctgccaggt atcaggcaag
     1621 gatatgggct cactgagact acatcagcta ttctgattac acccgagggg gatgataaac
     1681 cgggcgcggt cggtaaagtt gttccatttt ttgaagcgaa ggttgtggat ctggataccg
     1741 ggaaaacgct gggcgttaat caaagaggcg aactgtgtgt gagaggtcct atgattatgt
     1801 ccggttatgt aaacaatccg gaagcgacca acgccttgat tgacaaggat ggatggctac
     1861 attctggaga catagcttac tgggacgaag acgaacactt cttcatcgtt gaccgcctga
     1921 agtctctgat taagtacaaa ggctatcagg tggctcccgc tgaattggaa tccatcttgc
     1981 tccaacaccc caacatcttc gacgcaggtg tcgcaggtct tcccgacgat gacgccggtg
     2041 aacttcccgc cgccgttgtt gttttggagc acggaaagac gatgacggaa aaagagatcg
     2101 tggattacgt cgccagtcaa gtaacaaccg cgaaaaagtt gcgcggagga gttgtgtttg
     2161 tggacgaagt accgaaaggt cttaccggaa aactcgacgc aagaaaaatc agagagatcc
     2221 tcataaaggc caagaagggc ggaaagatcg ccgtgggatc catcgccacc atggtgagca
     2281 agggcgagga gctgttcacc ggggtggtgc ccatcctggt cgagctggac ggcgacgtaa
     2341 acggccacaa gttcagcgtg tccggcgagg gcgagggcga tgccacctac ggcaagctga
     2401 ccctgaagtt catctgcacc accggcaagc tgcccgtgcc ctggcccacc ctcgtgacca
     2461 ccctgaccta cggcgtgcag tgcttcagcc gctaccccga ccacatgaag cagcacgact
     2521 tcttcaagtc cgccatgccc gaaggctacg tccaggagcg caccatcttc ttcaaggacg
     2581 acggcaacta caagacccgc gccgaggtga agttcgaggg cgacaccctg gtgaaccgca
     2641 tcgagctgaa gggcatcgac ttcaaggagg acggcaacat cctggggcac aagctggagt
     2701 acaactacaa cagccacaac gtctatatca tggccgacaa gcagaagaac ggcatcaagg
     2761 tgaacttcaa gatccgccac aacatcgagg acggcagcgt gcagctcgcc gaccactacc
     2821 agcagaacac ccccatcggc gacggccccg tgctgctgcc cgacaaccac tacctgagca
     2881 cccagtccgc cctgagcaaa gaccccaacg agaagcgcga tcacatggtc ctgctggagt
     2941 tcgtgaccgc cgccgggatc actctcggca tggacgagct gtacaagtaa agcggccgcg
     3001 agcagtaatt ctagataagt ccaactacta aactggggat tcctgggccc tgaagaaggg
     3061 cccctcgact aagtccaact actaaactgg gccctgaaga agggcccata tagggccctg
     3121 aagaagggcc ctatcgagga tattatctcg aggactctag atcataatca gccataccac
     3181 atttgtagag gttttacttg ctttaaaaaa cctcccacac ctccccctga acctgaaaca
     3241 taaaatgaat gcaattgttg ttgttaactt gtttattgca gcttataatg gttacaaata
     3301 aagcaatagc atcacaaatt tcacaaataa agcatttttt tcactgcatt ctagttgtgg
     3361 tttgtccaaa ctcatcaatg tatcttaagg cgtaaattgt aagcgttaat attttgttaa
     3421 aattcgcgtt aaatttttgt taaatcagct cattttttaa ccaataggcc gaaatcggca
     3481 aaatccctta taaatcaaaa gaatagaccg agatagggtt gagtgttgtt ccagtttgga
     3541 acaagagtcc actattaaag aacgtggact ccaacgtcaa agggcgaaaa accgtctatc
     3601 agggcgatgg cccactacgt gaaccatcac cctaatcaag ttttttgggg tcgaggtgcc
     3661 gtaaagcact aaatcggaac cctaaaggga gcccccgatt tagagcttga cggggaaagc
     3721 cggcgaacgt ggcgagaaag gaagggaaga aagcgaaagg agcgggcgct agggcgctgg
     3781 caagtgtagc ggtcacgctg cgcgtaacca ccacacccgc cgcgcttaat gcgccgctac
     3841 agggcgcgtc aggtggcact tttcggggaa atgtgcgcgg aacccctatt tgtttatttt
     3901 tctaaataca ttcaaatatg tatccgctca tgagacaata accctgataa atgcttcaat
     3961 aatattgaaa aaggaagagt cctgaggcgg aaagaaccag ctgtggaatg tgtgtcagtt
     4021 agggtgtgga aagtccccag gctccccagc aggcagaagt atgcaaagca tgcatctcaa
     4081 ttagtcagca accatagtcc cgcccctaac tccgcccatc ccgcccctaa ctccgcccag
     4141 ttccgcccat tctccgcccc atggctgact aatttttttt atttatgcag aggccgaggc
     4201 cgcctcggcc tctgagctat tccagaagta gtgaggaggc ttttttggag gcctaggctt
     4261 ttgcaaagat cgatcaagag acaggatgag gatcgtttcg catgattgaa caagatggat
     4321 tgcacgcagg ttctccggcc gcttgggtgg agaggctatt cggctatgac tgggcacaac
     4381 agacaatcgg ctgctctgat gccgccgtgt tccggctgtc agcgcagggg cgcccggttc
     4441 tttttgtcaa gaccgacctg tccggtgccc tgaatgaact gcaagacgag gcagcgcggc
     4501 tatcgtggct ggccacgacg ggcgttcctt gcgcagctgt gctcgacgtt gtcactgaag
     4561 cgggaaggga ctggctgcta ttgggcgaag tgccggggca ggatctcctg tcatctcacc
     4621 ttgctcctgc cgagaaagta tccatcatgg ctgatgcaat gcggcggctg catacgcttg
     4681 atccggctac ctgcccattc gaccaccaag cgaaacatcg catcgagcga gcacgtactc
     4741 ggatggaagc cggtcttgtc gatcaggatg atctggacga agagcatcag gggctcgcgc
     4801 cagccgaact gttcgccagg ctcaaggcga gcatgcccga cggcgaggat ctcgtcgtga
     4861 cccatggcga tgcctgcttg ccgaatatca tggtggaaaa tggccgcttt tctggattca
     4921 tcgactgtgg ccggctgggt gtggcggacc gctatcagga catagcgttg gctacccgtg
     4981 atattgctga agagcttggc ggcgaatggg ctgaccgctt cctcgtgctt tacggtatcg
     5041 ccgctcccga ttcgcagcgc atcgccttct atcgccttct tgacgagttc ttctgagcgg
     5101 gactctgggg ttcgaaatga ccgaccaagc gacgcccaac ctgccatcac gagatttcga
     5161 ttccaccgcc gccttctatg aaaggttggg cttcggaatc gttttccggg acgccggctg
     5221 gatgatcctc cagcgcgggg atctcatgct ggagttcttc gcccacccta gggggaggct
     5281 aactgaaaca cggaaggaga caataccgga aggaacccgc gctatgacgg caataaaaag
     5341 acagaataaa acgcacggtg ttgggtcgtt tgttcataaa cgcggggttc ggtcccaggg
     5401 ctggcactct gtcgataccc caccgagacc ccattggggc caatacgccc gcgtttcttc
     5461 cttttcccca ccccaccccc caagttcggg tgaaggccca gggctcgcag ccaacgtcgg
     5521 ggcggcaggc cctgccatag cctcaggtta ctcatatata ctttagattg atttaaaact
     5581 tcatttttaa tttaaaagga tctaggtgaa gatccttttt gataatctca tgaccaaaat
     5641 cccttaacgt gagttttcgt tccactgagc gtcagacccc gtagaaaaga tcaaaggatc
     5701 ttcttgagat cctttttttc tgcgcgtaat ctgctgcttg caaacaaaaa aaccaccgct
     5761 accagcggtg gtttgtttgc cggatcaaga gctaccaact ctttttccga aggtaactgg
     5821 cttcagcaga gcgcagatac caaatactgt tcttctagtg tagccgtagt taggccacca
     5881 cttcaagaac tctgtagcac cgcctacata cctcgctctg ctaatcctgt taccagtggc
     5941 tgctgccagt ggcgataagt cgtgtcttac cgggttggac tcaagacgat agttaccgga
     6001 taaggcgcag cggtcgggct gaacgggggg ttcgtgcaca cagcccagct tggagcgaac
     6061 gacctacacc gaactgagat acctacagcg tgagctatga gaaagcgcca cgcttcccga
     6121 agggagaaag gcggacaggt atccggtaag cggcagggtc ggaacaggag agcgcacgag
     6181 ggagcttcca gggggaaacg cctggtatct ttatagtcct gtcgggtttc gccacctctg
     6241 acttgagcgt cgatttttgt gatgctcgtc aggggggcgg agcctatgga aaaacgccag
     6301 caacgcggcc tttttacggt tcctggcctt ttgctggcct tttgctcaca tgttctttcc
     6361 tgcgttatcc cctgattctg tggataaccg tattaccgcc atgcat
//
